First Report of Ranunculus white mottle ophiovirus in Slovenia in Pepper with yellow leaf curling symptom and in Tomato

Pepper (Capsicum annuum) and Tomato (Solanum lycopersicum) plants showing virus-like disease symptoms were collected in 2017, 2019, and 2020, in different parts of Slovenia (Supplementary Figure 1). Total RNA was extracted from leaf tissue of individual samples using RNeasy Plant Mini kit (Qiagen) and pooled in four composite samples as follows: 2 pepper plants from 2017 (D2017), 5 pepper and 4 tomato plants from 2019 (D2019_P1), 7 tomato plants (D2020_P1), and 2 pepper and 4 tomato plants (D2020_P3) from 2020. The pooled RNA samples were sequenced using Illumina platforms, details of the sequencing experiments are in Supplementary Table 1. Reads were analyzed using CLC Genomics Workbench (v. 20.0, Qiagen) following the pipelines for plant virus discovery (Pecman et al., 2017). Reads and contigs mapping to Ranunculus white mottle ophiovirus (RWMV, GenBank accession no. AY542957 or NC_043389) were detected in all pools. The longest contig (1,255 bp) was obtained from the 2019 composite sample, mapping to the coat protein-coding RNA 3 segment of the RWMV genome (accession no. AY542957). Details of mapping, genome coverage, and other viruses detected in the pools are summarized in the Supplementary Table 1. To identify individual RWMV-infected plants from the pools, primers were designed for detection by reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene (see Supplementary Table 2). Two pepper samples from two different farms, collected in 2017 and 2019 in southwest Slovenia, and four tomato samples from two different farms, collected in 2020 in central Slovenia tested positive for RWMV in RT-PCR assays. To assess the diversity of RWMV isolates, amplicons were purified using QIAquick PCR purification kit (Qiagen) and sent for Sanger sequencing. Based on maximum likelihood phylogenetic analysis, RWMV Italian and Slovenian isolates form a monophyletic clade within the genus (see Supplementary Figure 2). Pairwise nucleotide identities of the Slovenian isolates (accession no. MZ507604-MZ507609), relative to the original Italian isolate coat protein (accession no. AY542957) range from 92-97%, indicating a moderate level of diversity among isolates (see Supplementary Figure 2). Since only RWMV, bell pepper alphaendornavirus (BPEV), and pepper cryptic virus 2 (PepCV2), were present in a pepper sample from 2017, and BPEV and PepCV2 infection in pepper are not known to be associated with any of the disease symptoms (Okada et al., 2011; Saritha et al., 2016), the symptoms observed on this plant might be associated with RWMV infection. We observed mottling with interveinal chlorosis or yellowing, slight to full curling of leaves from lamina inward, as well as necrotic and aborted flowers on this plant (see Supplementary Figure 1). We cannot easily associate observed symptoms with RWMV in RWMV-positive tomatoes, since several viruses were detected in the pools containing these samples. Nevertheless, the prominent symptoms in tomato were mottling with interveinal chlorosis and leaf curling, similar to those observed in pepper. RWMV was discovered and characterized in buttercup (Ranunculus asiaticus), and detected in anemone (Anemone coronaria), from Italy (Vaira et al., 1996, 1997, 2000, 2003). It was recently detected in pepper from Australia showing veinal yellowing (Gambley et al., 2019). Here, we detected RWMV for the first time in Slovenia, and reported its first detection in tomato and pepper from Europe. These findings call for further studies on the effects of RWMV infection on tomato and pepper production, and its monitoring in neighboring European countries. Acknowledgment This study received funding from the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection, Slovenian Research Agency (ARRS) core financing (P4-0165), and the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-ITN) project “INEXTVIR” (GA 813542), under the management of the European Commission-Research Executive Agency. References Gambley, C., et al. 2019. New Dis. Rep. 40:13. doi:10.5197/j.2044-0588.2019.040.013. Okada, R., et al. 2011. J. Gen. Virol. 92:2664-2673. doi:10.1099/vir.0.034686-0. Pecman, A., et al. 2017. Front. Microbiol. 8:1-10. doi:10.3389/fmicb.2017.01998. Saritha, R. K., et al. 2016. VirusDisease 27:327-328. doi:10.1007/s13337-016-0327-7. Vaira, A. M., et al. 2003. Arch. Virol. 148:1037-1050. doi:10.1007/s00705-003-0016-x. Vaira, A. M., et al. 1996. Acta Hortic., 432:36-43. doi:10.17660/ActaHortic.1996.432.3. Vaira, A. M., et al. 1997. Arch. Virol. 142:2131-2146. doi:10.1007/s007050050231. Vaira, A. M., et al. 2000. Plant Dis. 84:1046-1046. doi:10.1094/PDIS.2000.84.9.1046B.

First report of peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and mume virus A (MuVA) from Mei (Prunus mume) in China

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5′-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5′-GCCAAAAGAAGTGCTGGTGG-3′ [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5′-AAGAGAATTACTTCAATGCCCTC-3′ [positions 171-194, NC_040568.1] / Reverse: 5′-GATATCCAAGATACGATTAGCCAG-3′ [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5′-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5′- GAGTGATACATAGTCCACAGAGAT-3′[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.

Response of Calceolaria ×herbeohybrida Cultivars to Substrate pH and Corresponding Leaf Tissue Nutrient Concentration Effects

Calceolaria (Calceolaria ×herbeohybrida) is a flowering potted greenhouse crop that often develops upper-leaf chlorosis, interveinal chlorosis, and marginal and leaf-tip necrosis (death) caused by cultural practices. The objectives of this research were to 1) determine the optimal incorporation rate of dolomitic and/or hydrated lime to increase substrate pH; 2) determine the influence of the liming material on substrate pH, plant growth, and leaf tissue nutrient concentrations; and 3) determine the optimal substrate pH to grow and maintain during calceolaria production. Sphagnum peatmoss was amended with 20% (by volume) perlite and incorporated with pulverized dolomitic carbonate limestone (DL) and/or hydrated limestone (HL) at the following concentrations: 48.1 kg·m−3 or 144.2 kg·m−3 DL, 17.6 kg·m−3 DL + 5.3 kg·m−3 HL, or 17.6 kg·m−3 DL + 10.6 kg·m−3 HL to achieve a target substrate pH of 4.5, 5.5, 6.5, and 7.5, respectively. Calceolaria ‘Orange’, ‘Orange Red Eye’, ‘Yellow’, and ‘Yellow Red Eye’ were grown in each of the prepared substrates. For all cultivars, substrate solution pH increased as limestone incorporation concentration and weeks after transplant (WAT) increased, although to different magnitudes. For example, as limestone incorporation increased from 48.1 kg·m−3 DL to 17.6 kg·m−3 DL + 10.6 kg·m−3 HL, substrate solution pH for ‘Orange’ calceolaria increased from 4.1 to 6.9 to 4.8 to 7.2 at 2 and 6 WAT, respectively. Substrate solution electrical conductivity (EC) and growth indices were not influenced by limestone incorporation, but total plant dry mass increased. Few macronutrients and most micronutrients were influenced by limestone incorporation. Leaf tissue iron concentrations for ‘Orange’, ‘Orange Red Eye’, ‘Yellow’, and ‘Yellow Red Eye’ calceolaria decreased by 146%, 91%, 71%, and 84%, respectively, when plants were grown in substrates incorporated with increasing limestone concentrations from 144.2 kg·m−3 DL to 17.6 kg·m−3 DL + 10.6 kg·m−3 HL (pH 6.5–6.9). Therefore, incorporating 144.2 kg·m−3 DL into peat-based substrates and maintaining a pH <6.5 will avoid high pH–induced Fe deficiency and prevent upper-leaf and interveinal chlorosis.

Magnesium-Deficiency Effects on Pigments, Photosynthesis and Photosynthetic Electron Transport of Leaves, and Nutrients of Leaf Blades and Veins in Citrus sinensis Seedlings

Citrus sinensis seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg (NO3)2 for 16 weeks. Mg-deficiency-induced interveinal chlorosis, vein enlargement and corkiness, and alterations of gas exchange, pigments, chlorophyll a fluorescence (OJIP) transients and related parameters were observed in middle and lower leaves, especially in the latter, but not in upper leaves. Mg-deficiency might impair the whole photosynthetic electron transport, including structural damage to thylakoids, ungrouping of photosystem II (PSII), inactivation of oxygen-evolving complex (OEC) and reaction centers (RCs), increased reduction of primary quinone electron acceptor (QA) and plastoquinone pool at PSII acceptor side and oxidation of PSI end-electron acceptors, thus lowering energy transfer and absorption efficiency and the transfer of electrons to the dark reactions, hence, the rate of CO2 assimilation in Mg-deficiency middle and lower leaves. Although potassium, Mg, manganese and zinc concentration in blades displayed a significant and positive relationship with the corresponding element concentration in veins, respectively, great differences existed in Mg-deficiency-induced alterations of nutrient concentrations between leaf blades and veins. For example, Mg-deficiency increased boron level in the blades of upper leaves, decreased boron level in the blades of lower leaves, but did not affect boron level in the blades of middle leaves and veins of upper, middle and lower leaves. To conclude, Mg-deficiency-induced interveinal chlorosis, vein enlargement, and corkiness, and alterations to photosynthesis and related parameters increased with increasing leaf age. Mg-deficiency-induced enlargement and corkiness of veins were not caused by Mg-deficiency-induced boron-starvation.

Foliar Application of Dikegulac Sodium Increases Branching of ‘Merritt’s Supreme’ Bigleaf Hydrangea

The goal of this experiment was to evaluate the efficiency of foliar application of dikegulac sodium on increasing the lateral branching of ‘Merritt’s Supreme’ bigleaf hydrangea (Hydrangea macrophylla). Plants were grown in greenhouses at two locations including El Paso, TX and Kosciusko, MS. Two weeks before application of dikegulac sodium, half of plants were hand-pinched leaving two nodes. Foliar spray of dikegulac sodium at 400, 800, or 1600 mg·L−1 was then applied to pinched and unpinched plants. There were two additional control treatments: pinched or unpinched without application of dikegulac sodium. Data were collected at 2 weeks, 6 weeks, 80 days, and 10 months after treatments. Bigleaf hydrangea plants exhibited severe phytotoxicity including interveinal chlorosis or bleaching of new growth at 2 weeks after application of dikegulac sodium with more pronounced symptoms at higher dikegulac sodium concentrations. The severity of phytotoxicity symptoms became less significant at 6 weeks after treatment. The effect of dikegulac sodium on bigleaf hydrangea plant growth, number of branches, and number of flowers depended on both locations and dosages. In El Paso, TX, dikegulac sodium at 800 or 1600 mg·L−1 inhibited bigleaf hydrangea plant growth at 6 weeks and 80 days after treatment, and this effect disappeared at 10 months after treatment. Dikegulac sodium at all tested dosages doubled or tripled the number of branches of pinched or unpinched bigleaf hydrangea, respectively, at 80 days after treatment. At 10 months after treatment, the number of branches and flowers of bigleaf hydrangea plants tended to increase, but was insignificant. In Kosciusko, MS, dikegulac sodium at 1600 mg·L−1 reduced the plant growth at 6 weeks after treatment. This treatment increased the number of branches and flowers of unpinched plants by 196% and 95% and pinched plants by 53% and 31%, respectively, at 10 months after treatment. Dikegulac sodium application could be used to increase number of branches and flowers and produce compact ‘Merritt’s Supreme’ bigleaf hydrangea. However, the efficacy varied with environmental conditions.

First Report of Tomato chlorosis virus in Potato in Brazil

Potato plants (Solanum tuberosum cv. Ágata) exhibiting symptoms of leaf roll and interveinal chlorosis, especially on older leaves, were found in a commercial crop in the County of Cristalina, State of Goiás, Brazil in June 2011. The crop was severely infested by whitefly Bemisia tabaci biotype B. Four potato tubers from symptomatic plants were indexed for the presence of the following viruses: Tomato chlorosis virus (ToCV), Potato leaf roll virus (PLRV), Tomato severe rugose virus (ToSRV), and Potato virus Y (PVY). Total RNA was extracted separately from each tuber and used for reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for ToCV. The RT-PCR product was subsequently tested by nested-PCR for detection of ToCV with specific primers ToC-5/ToC-6 (2). Amplicons of 463 bp, amplified from total RNA separately extracted from three tubers, were purified and directly sequenced. Comparisons among the three consensus sequences of 448 bp (GenBank Accession Nos. JQ288896, JQ288897, and JQ288898) revealed respectively, 98, 100, and 100% identity with the reported sequence of a tomato isolate of ToCV from Brazil (GenBank Accession No. EU868927) (1). For ToSRV detection, total DNA was extracted from two tubers and a fragment of approximately 820 bp was amplified by PCR with specific primers (3). PLRV and PVY were indexed in two and three tubers, respectively, by double-antibody sandwich-ELISA (SASA, Edinburg, Scotland). Virus-free B. tabaci biotype B were separately transferred to potato and tomato leaves infected with ToCV for an acquisition access period of 24 h. Groups of 30 viruliferous whitefly were transferred to four, young, sprout-grown potato plants cv. Ágata (two plants per virus isolate) for 24-h inoculation access period. After 37 days of inoculation, one plant inoculated with the potato and tomato isolates of ToCV, respectively exhibited symptoms of leaf roll and interveinal chlorosis on order leaves, which were similar to that induced by PLRV. Experimental infection of potato plants with ToCV, which induced leaf roll symptoms resembling PLRV infection, was reported in the United States by Wisler et al. (4). The potato isolate of ToCV was also transmitted by B. tabaci to one of two inoculated tomato plants. The presence of ToCV in all inoculated plants was detected by nested-RT-PCR as described above. To our knowledge, this is the first report on detection of ToCV in field potato plants in the world. Considering that ToCV occurs in innumerous countries around the world, it is transmitted by a cosmopolitan insect, and it induces symptoms similar to PLRV, this finding triggers an alert to field dependent seed-potato multiplication, virus inspector, and certification system. References: (1) J. C. Barbosa et al. Plant Dis. 92:1709, 2008. (2) C. I. Dovas et al. Plant Dis. 86:1345, 2002. (3) F. R. Fernandes et al. Trop. Plant Pathol. 35:43, 2010. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.

First Report of Tomato chlorosis virus Infecting Tomato in Georgia

Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV) are prevalent in field-grown tomato (Solanum lycopersicum) production in Georgia. Typical TYLCV symptoms were observed during varietal trials in fall 2009 and 2010 to screen genotypes against TYLCV at the Coastal Plain Experiment Station, Tifton, GA. However, foliar symptoms atypical of TYLCV including interveinal chlorosis, purpling, brittleness, and mottling on upper and middle leaves and bronzing and intense interveinal chlorosis on lower leaves were also observed. Heavy whitefly (Bemisia tabaci (Gennadius), B biotype) infestation was also observed on all tomato genotypes. Preliminary tests (PCR and nucleic acid hybridization) in fall 2009 indicated the presence of TYLCV, TSWV, Cucumber mosaic virus, and Tomato chlorosis virus (ToCV); all with the exception of ToCV have been reported in Georgia. Sixteen additional symptomatic leaf samples were randomly collected in fall 2010 and the preliminary results from 2009 were used to guide testing. DNA and RNA were individually extracted using commercially available kits and used for PCR testing for ToCV, TYLCV, and TSWV. Reverse transcription (RT)-PCR with ToCV CP gene specific primers (4) produced approximately 750-bp amplicons from nine of the 16 leaf samples. Four of the nine CP gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 100.0% identical with each other (GenBank Accession Nos. HQ879840 to HQ879843). They were 99.3 to 99.5%, 97.2 to 97.5%, and 98.6 to 98.9% identical to ToCV CP sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. The presence of ToCV was confirmed by amplifying a portion of the HSP70h gene using the primers HSP-1F and HSP-1R (1). RT-PCR produced approximately 900-bp amplicons in the same nine samples. Four HSP70h gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 99.7% identical to each other (Accession Nos. HQ879844 to HQ879847). They were 99.2 to 99.5%, 98.0 to 98.4%, and 98.9 to 99.3% identical to HSP70h sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. TYLCV was also detected in all 16 samples by PCR using degenerate begomovirus primers PAL1v 1978 and PARIc 496 (3) followed by sequencing. TSWV was also detected in two of the ToCVinfected samples by RT-PCR with TSWV N gene specific primers (2) followed by sequencing. To our knowledge, this is the first report of the natural occurrence of ToCV in Georgia. Further studies are required to quantify the yield losses from ToCV alone and synergistic interactions between ToCV in combination with TSWV and/or TYLCV in tomato production in Georgia. References: (1) T. Hirota et al. J. Gen. Plant Pathol. 76:168, 2010. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) L. Segev et al. Plant Dis. 88:1160, 2004.